PROJECT SUMMARY Several studies link mental health problems such as major depression, bipolar disorder and schizophrenia with G-protein coupled receptors (GPCRs), including adrenergic, dopaminergic and cholinergic receptors. Simultaneously, there are more than 140 GPCRs identified through the human genome project where their endogenous ligand is still unknown. In search of these ligands, there is an emerging need for methods that can provide detailed information of ligand affinity and their putative cellular response. Investigations of ligand? receptor interactions should ideally take place as close as possible to the native state of a biological cell with minimal perturbation. Here, we propose to develop a novel probe of cell function, based on ambient sampling mass spectrometry (MS), where ligand?receptor interactions can be monitored in situ by forming a liquid junction between two fused silica capillaries and immersing them on top of a surface with cells acting as a biological model for neurotransmission. The first capillary acts as a perfusion system, the second capillary pulls on the liquid junction and delivers a spray of analytes at the MS-inlet for detection. The capability of measuring a ligand-receptor binding event, the subsequent release of neurotransmitters and any enzymatic conversion of the released molecules taking place on the cell surface is made possible by the multiplexing abilities of MS. The technique does not require derivatization to detect analytes and is non-invasive. Using the PC12 cell line, the affinity of several classic neurotransmitters will be screened against endogenous muscarinic receptors as a proof-of-concept for this novel method. The technique will also be used to perform measurements of cellular responses such as release of dopamine from PC12 cells. The establishment of the proposed technique is highly translational in the sense that it may be applied to virtually any type of tissue and ligand-receptor interaction. Understanding the complexity of cellular mechanisms in neurons is a challenging task as recognized by the BRAIN initiative and ligand-receptor interactions are a fundamental part of cell-to-cell signaling. The proposed groundbreaking research will develop a method for measuring ligand?receptor interactions on the surface of a cell using mass spectrometry. To detect and measure these events with ambient sampling coupled to MS has hitherto never been done. This is a challenging yet possible task that will be achieved through collaboration between the Zare group at Stanford University and the Zhong group at Central China Normal University. The project combines recent advances of ambient sonic spray ionization with classic microperfusion, nanoflow liquid chromatography and high-resolution MS, and is expected to interrogate ligand?receptor interactions on live cells.